WO2012168065A1 - High-resolution luminescence microscopy - Google Patents

Abstract

The invention relates to a microscopy method for generating a high-resolution image (55) of a sample (2), the method comprising the following steps: a) the sample (2) is provided with a marker, which upon excitation emits statistically blinking luminescent radiation, or a sample (2) is used which upon excitation emits locally distributed, statistically blinking luminescent radiation; b) the sample (2) is excited to luminescence by means of structured illumination, wherein the sample is repeatedly illuminated in at least nine different illumination conditions (.01-.09) of the structured illumination by realizing at least three rotary positions, and at least three displacement positions per rotary position of the structured illumination; c) the luminescing sample (2) is repeatedly displayed in each of the different illumination conditions on a flat panel detector having pixels, such that an image sequence (44.01-44.09) is obtained for each of the different illumination conditions (.01-.09); d) a raw image, the spatial resolution of which exceeds the optical resolution of the image is generated from each image sequence (44.01-44.09) by means of a cumulant function (46), which evaluates intensity fluctuations of each pixel in the image sequence, said intensity fluctuations being caused by the blinking, so that for each of the different illumination conditions (.01-.09), a high-resolution raw image (53.01-53.09) is obtained; e) from the raw images (53.01-53.09) thus obtained, the high-resolution image (55), which relative to the raw images (53.01-53.09) has an increased spatial resolution, is generated by way of computational processing (42) comprising Fourier filtering.

Description

 High-resolution luminescence microscopy

The invention relates to a microscopy method or a microscope for generating a high-resolution image of a luminescent sample.

The examination of samples by microscopy is a wide technical field, for which there are many technical solutions. Starting from classical light microscopy, various microscopy techniques have developed. A classical field of application of light microscopy for the investigation of biological preparations is luminescence microscopy. Here, certain dyes (so-called phosphors or fluorophores) are used for the specific labeling of samples, e.g. of cell parts, used. As mentioned, the sample is illuminated with excitation radiation and the luminescence light stimulated thereby is detected with suitable detectors. Typically, a dichroic beam splitter in combination with block filters is provided for this purpose in the light microscope, which split off the fluorescence radiation from the excitation radiation and enable separate observation. By this procedure, the representation of individual, differently colored cell parts in the light microscope is possible. Of course, several parts of a preparation can be colored simultaneously with different, specifically attaching to different structures of the preparation dyes. This process is called multiple luminescence. It is also possible to measure samples that luminesce per se, ie without the addition of marker.

Luminescence is understood here, as is common practice, as a generic term for phosphorescence and fluorescence, so it covers both processes.

Furthermore, it is known for sample examination to use laser scanning microscopy (also abbreviated LSM), which from a three-dimensionally illuminated image by means of a confocal detection arrangement (one speaks of a confocal LSM) or a nonlinear sample interaction (so-called multiphoton microscopy) only the one Plain level, which is located in the focal plane of the lens. An optical section is obtained, and the recording of several optical sections at different depths of the sample then makes it possible, with the aid of a suitable data processing device, to generate a three-dimensional image of the sample, which is composed of the various optical sections. Laser scanning microscopy is thus suitable for the examination of thick specimens.

For resolutions beyond the diffraction limit given by the laws of physics, various approaches have recently been developed. These microscopy methods are characterized in that they provide the user with a higher lateral and / or axial optical resolution compared to the conventional microscope. In this specification, such microscopy methods are referred to as high-resolution microscopy methods, since they achieve a resolution beyond the optical diffraction limit. Diffraction-limited microscopes, on the other hand, are called classical microscopes. They realize known optical far field microscopy or laser scanning microscopy.

A high-resolution microscopy method is mentioned in EP 1 157 297 B1. In the process, nonlinear processes are exploited by means of structured illumination. The non-linearity is the saturation of the fluorescence. Structured illumination results in a displacement of the object space spectrum relative to the transfer function of the optical system. Specifically, the shift of the spectrum means that object space frequencies V0 are transmitted at a spatial frequency V0-Vm, where Vm is the frequency of the structured illumination. Given a maximum spatial frequency that can be transmitted by the system, this enables the transfer of spatial frequencies of the object lying around the shift frequency Vm above the maximum frequency of the transfer function. This approach requires Fourier filtering as a reconstruction algorithm for image generation and exploitation of multiple images for an image. The EP 1 157 297 B1, which is also fully incorporated with regard to the corresponding description of the resolving microscopy method, thus uses a structured far-field illumination of the sample, for example by means of an amplitude / phase grating. Fluorescence in the sample is also detected far field. The grid is now used in at least three different rotational positions, z. B. 0 °, 120 ° and 240 °, and in each rotational position, the grid is moved in three or more different positions. For the three shifts of the three rotational positions (a total of at least 9 illumination states), the sample is detected far field. Furthermore, the grating has frequencies as close as possible to the cut-off frequency, to the transmission of which the optical arrangement used is capable. Using the Fourier filtering then the mentioned shift, in particular the 0. and +/- 1. Diffraction order is evaluated in the images. This microscopy method is also called a SIM method. An increase of the resolution is obtained with this principle, if the structuring (eg by a grid) is so intense that the fluorescence of the sample reaches saturation in the bright area. Then the structured illumination of the sample on the sample no longer has a sinusoidal distribution, but because of the saturation effects even higher harmonic beyond the optical cutoff frequency. This development of the SIM method is also referred to as saturated pattern excitation microscopy (SPEM).

A further development of the SIM method can also be achieved with a linear illumination which is perpendicular to the grid direction. You then have a line lighting, along the line, the grid structure is found again. The lines of the lighting is thus in turn structured by the grid. The linear illumination allows a confocal slit detection and thus an increase in resolution. This procedure is also abbreviated SLIM.

The paper C. Müller and J. Enderlein, "Image scanning microscopy", Physical Review Letters, 104, 198101 (2010) uses the SIM principle, but uses a scanning of the sample with a confocal illumination and detection with subsequent Fourier filtering The principle is called ISM, which does not have nine orientations of structured illumination, but each scan position, ie each raster state when the image is scanned, corresponds to a lighting state, and the structured illumination is spot illumination of the sample.

From the publication T. Dertinger, et al., "Fast, background-free, 3D super-resolution optical fluctuation imaging (SOFI)", PNAS (2009), pp 22287-22292 and "Achieving increased resolution and more pixels with superresolution Optical Fluctuation Imaging (SOFI), Opt. Express, 30.08.2010, 18 (18): 18875-85, doi: 10.1364 / 1 E.18.018875 and S. Geissbuehler et al., "Comparison between SOFI and STORM", Biomed Opt. Express 2, 408-420 (201 1) discloses another high-resolution method of luminescence microscopy that utilizes the blinking properties of a fluorophore: If the fluorophores of a sample flash statistically independently of each other, the sample can be imaged by appropriate filtering with a so-called "fluorescence" microscope An example of such a cumulant function is, for example, the second order autocorrelation function g. To produce a high-resolution image, a sequence of individual images is recorded and then combined with the cumulant function to form a single image, which then has the higher resolution. This method is abbreviated to the term "Super-Resolution Optical Fluctuation Imaging" as the SOFI method. It is further known in the prior art to combine several high-resolution microscopy methods. For example, DE 102008054317 A1 describes the combination of various high-resolution microscopy methods, with the aim of being able to use the optimum method for individual sample areas in each case from the viewpoints of resolution and measuring speed.

The invention has for its object to provide a microscopy method or a microscope, which achieves an increased resolution.

This object is achieved according to the invention with a microscopy method for producing a high-resolution image of a sample, the method comprising the following steps: a) the sample is provided with a marker which emits statistically flashing luminescence radiation after excitation or a sample is used which after excitation b) the sample is excited to luminescence by structured illumination, the sample being repeatedly illuminated in different illumination states of the structured illumination, c) the luminescent sample is repeatedly imaged onto a detector in each of the different illumination states, so that a sequence of images is obtained for each of the different illumination states, d) from each image sequence by means of a cumulant function, which evaluates intensity fluctuations caused by the flashing in the image sequence, a raw image which has a di e) an increased resolution of the spatial resolution is generated so that a high-resolution raw image is obtained for each of the different illumination states; e) from the raw images obtained by a computational processing comprising a Fourier filtering the high-resolution image which has a spatial resolution which is increased compared to the individual images has generated.

The object is further achieved according to the invention with a microscopy method for producing a high-resolution image of a sample, the method comprising the following steps: a) the sample is provided with a marker which emits statistically flashing luminescence radiation after excitation, or a sample is used which after Localized excitation, statistically flashing emitting luminescent radiation, b) the sample is excited by luminescence by structured illumination, the sample being repeatedly illuminated with a set of different illumination states of the structured illumination, c) the luminescent sample in each set and there in each of the sets mapped different illumination states to a detector, so that a sequence of sentences, each comprising an image per different illumination state is obtained, d) from each set by a Fourier filtering comprehensive arithmetic processing a raw image with the optis che E) from the sequence of raw images by means of a cumulant function, which evaluates intensity fluctuations in the sequence caused by the flashing, the high-resolution image, which is one compared to the raw images, is generated Spatial resolution has generated.

The object is finally also solved with a microscope for generating a high-resolution image of a sample, the sample is provided with a marker that emits statistically flashing luminescence after excitation, or a sample that distributes locally after excitation, flashing emits statistically flashing luminescence, wherein the Microscope comprising: a detection beam path and an illumination beam path, wherein the detection beam path images the sample detector and the illumination beam path illuminates the sample with structured illumination for excitation of luminescence, wherein the illumination beam path comprises means for realizing different illumination states, a computing and control device, the controlling the microscope so that the sample is repeatedly illuminated to the different illumination states of the structured illumination, repeating the luminescent sample in each of the different illumination states is imaged on a detector, so that an image sequence is obtained for each of the different illumination states, the arithmetic and control device from each image sequence by means of a cumulant function, which evaluates caused by the flashing intensity fluctuations in the image sequence, a raw image, the one on the optical resolution the image has increased spatial resolution generated so that a high-resolution raw image is obtained for each of the different lighting conditions, the arithmetic and control device from the raw images obtained by a Fourier filtering comprehensive arithmetic processing, the high-resolution image having a relation to the individual images increased spatial resolution , generated.

The object is finally solved with a microscope for generating a high-resolution image of a sample, the sample is provided with a marker that emits statistical flashing luminescence after excitation, or a sample that distributes locally after excitation, statistically flashing emitting luminescence, wherein the Microscope comprising: a detection beam path and an illumination beam path, wherein the detection beam path images the sample detector and the illumination beam path illuminates the sample with structured illumination for excitation of luminescence, wherein the illumination beam path comprises means for realizing different illumination states, a computing and control device, the controlling the microscope so that the sample is repeatedly illuminated with a set of different illumination states of the structured illumination, and the luminescent sample in each set and is there imaged onto the detector in each of the different illumination conditions, so that a sequence of sets each comprising one image per different illumination state is obtained, the arithmetic and control means from each set a computational processing comprising a Fourier filtering including a raw image generates the optical resolution of the image of increased spatial resolution, so that a sequence of raw images is obtained, the arithmetic and control means from the sequence of raw images by means of a cumulant function, which evaluates intensity fluctuations in the sequence caused by the flashing, the high-resolution image, the compared to the raw images increased spatial resolution generated.

According to the invention, elements of the SIM / ISM principle are combined with elements of the SOFI principle. A clever combination achieves an increase in resolution that is better than the factor 2, which would result from the combination of the individual high-resolution images. The image is resolved higher than it would allow the averaging of images of the individual process characteristics at the same effort.

When using the SIM principle, the sample is illuminated in at least nine different illumination states of the structured illumination by implementing at least three rotational positions and at least three shift positions of the structured illumination per rotational position. The luminescent sample is repeatedly imaged onto a pixel-containing area detector in each of the different illumination states. The intensity fluctuations in the sequence caused by the flashing are evaluated for each pixel. When using the ISM principle, the illumination conditions are the different scan positions that occur during scanning. Unlike a conventional LSM, the images of the sample in the different illumination states have a resolution showing the structure of the fluorescent elements. This can be z. B. by a corresponding resolution wide field detection.

The images of the fluorescent sample obtained at the different illumination conditions then represent a set of images. The acquisition of such sets is repeated to obtain a sequence of sentences. From this sequence of sentences, a high-resolution image can now be obtained in two ways:

1 . Each set can be converted to a high-resolution raw image using the computational processing known for SIM / ISM using Fourier filtering. One then obtains a sequence of raw images in this way. This episode of Raw images are then converted to a still high-resolution final image using a cumulant function according to the SOFI approach. The resolution of the final image is significantly increased over that of the raw image, in particular one obtains a result in the result, which would be much better than a simple aggregation of images, which with the principles SIM or

SOFI were won.

2. However, the sequence of sentences, each comprising an image of the sample in the different lighting conditions, may also be determined first for each SOFI lighting state using the

Cumulant function are converted into a set of raw images containing a raw image for each lighting condition. This set of raw images can then be converted by means of the SIM / ISM-like approach into a final, also further increased in its resolution image.

Since the invention includes features of the SIM principle, its following references, which deal with details of the SIM principle, fully incorporated in its disclosure here: EP 1 157 297 B1, DE 19908883 A1, M. Gustafsson, "Nonlinear structured-illumination microscopy : Wide-field fluorescence imaging with theoretically unlimited resolution ", Proc Natl Acad Sci USA, 13.09.2005, 102 (37): 13081 -6, Epub 2.09.2005, R. Heintzmann and C. Cremer, (1998), Proc. Soc., Opt., Eng., 3568, pp. 185-195, The same applies to the ISM publication C. Müller and J. Enderlein, "Image scanning microscopy," Physical Review Letters, 104, 198101 (2010). Since the invention also uses features of the SOFI principle, the following publications relevant for this principle are also fully integrated: T. Dertinger et al., "Fast, background-free, 3D super-resolution optical fluorescence imaging (SOFI)", PNAS (2009), p. 22287-22292 and associated "Supporting Information"; "Optically Expressing, 30.08.2010, 18 (18): 18875-85, doi: 10.1364 / IE.18.018875 and S. Geissbuehler et al.," Comparison between SOFI and STORM ", Biomed. Opt. Express 2, pp. 408-420 (201 1).

According to the invention, the sample is provided with a marker which emits statistically flashing luminescent radiation after suitable excitation. Under markers thereby usual label or other substances are provided, which attach themselves to represented structures of the sample. Alternatively, an already suitably luminescent sample can be used. Ultimately, therefore, a sample is microscopically imaged, which has structures that emit statistically flashing luminescent. "Statistically flashing" means that the individual markers or sample molecules in each case independently of each other of adjacent markers or molecules constantly change between two luminescence radiation states. In the simplest case, this may be a state in which luminescence radiation is emitted and a state in which no luminescence radiation is emitted. But it is also the change between the delivery of two different types of luminescence possible, for example, a wavelength change, polarization change, etc. This property of the sample is necessary for the application of the elements from the SOFI principle, since only then the cumulant function allows one To convert image sequence into a single high-resolution image.

If one uses the above-mentioned second variant in which a sequence of sets of images of the sample in different illumination states by Fourier transformation into a sequence of higher-resolution raw images is converted, the flashing behavior within a sentence should not change as possible. This means that the time period in which a sentence is recorded should be short in comparison with the mean flashing frequency, ie should not be longer than the reciprocal of the flashing frequency or particularly preferably a fraction of the reciprocal. This corresponds to the feature that the sentence rate is smaller, preferably 10 times smaller than the average frame rate. Then it is ensured that the blinking state of the molecules involved does not change within a sentence. This is an advantage for the SOFI processing executed on the sequence of raw images.

It is therefore preferred in a development of the invention that the image acquisition rate and the blink rate of the molecules used are suitably adapted to one another. This can be achieved by selecting suitable marker or sample molecules. Of course it is also possible to adjust the blink rate of the molecules by appropriate influence. Depending on the molecule, different physical influencing variables come into question here, in particular temperature, wavelength of the illumination radiation acting as excitation radiation, intensity of the illumination radiation acting as excitation radiation, etc. Also, chemical influences are possible, such. From Geissbuehler et al. explained. Further, for a given blink rate, one can adjust the frame rate (rate of frames with pictures) accordingly to achieve the above-mentioned condition.

As far as the invention is described above or below with reference to process features, this should equally apply to the description of a corresponding microscope from the above-mentioned features. Essential for the microscope is that its computing and control device ensures a corresponding operation of the microscope for the realization of the described process characteristics. The computational and Control device is designed to suit, for example, by a suitable programming. The opposite applies, of course, in the event that individual features should be described only on the basis of the microscope. These features then apply analogously to the described microscopy methods.

For SIM, SLIM and SPEM a rotation of the structured illumination is required, as already described. This can be realized in a particularly simple manner, if the grating, which lies to realize this microscopy method in the common section of the second and third illumination beam path, is followed by an image field rotator. This can be realized for example by an Abbe-King prism. This subordinate Bildfeldrotator is particularly advantageous for the SLIM principle, since then the vertical position of grid and line lighting must be set only once ß and tracking, which would otherwise be required in a lattice rotation, is eliminated. It is understood that the features mentioned above and those yet to be explained below can be used not only in the specified combinations but also in other combinations or alone, without departing from the scope of the present invention. The invention will be explained in more detail for example with reference to the accompanying drawings, which also disclose characteristics essential to the invention. Show it :

1 shows a schematic representation of a combination microscope,

 Fig. 2 is a schematic representation to illustrate the generation of a high-resolution

Image from several wide field images of a sample under different

Lighting conditions were obtained,

Fig. 3 is a schematic illustration for illustrating the generation of a high-resolution

Image from a sequence of images of the sample in different flashing states below

Combination of the principles according to FIG. 2 and FIG. 3,

4 shows a first embodiment of a method for generating an even higher-resolution image with combination of the principles according to FIGS. 2 and 3 and FIG

5 shows a second embodiment of a method for generating a likewise higher-resolution image likewise with combination of the principles according to FIG

FIGS. 2 and 3.

In Fig. 1, a microscope 1 is shown, the classical microscopy method, d. H . Microscopy method whose resolution diffraction-limited, with high-resolution Simultaneously perform microscopy method, ie with microscopy method whose resolution is increased beyond the diffraction limit.

The microscope 1 is constructed on the basis of a conventional laser scanning microscope and detects a sample 2. For this purpose, it has an objective 3, through which the radiation runs for all microscopy methods.

The objective 3 forms, via a beam splitter 4, the specimen together with a tube lens 5 on a CCD detector 6, which in the example is a generally possible area detector. In this respect, the microscope 1 has a conventional light microscope module 7 and the beam path from the sample 2 through the lens 3 and the tube lens 5 to the CCD detector 6 corresponds to a conventional wide-field detection beam path 8. The beam splitter 4, as indicated by the double arrow in FIG. 1, interchangeable, in order to be able to switch between beam splitters with different dichroic properties or achromatic beam splitters according to US 2008/0088920.

In the beam path to the lens 3, a laser scanning module 9 is further connected, whose LSM illumination and detection beam path via a switching mirror 1 1, which also has beam splitter functions, is coupled into the beam path to the lens 3. The beam path from the switching mirror 1 1 to the lens 3 through the beam splitter 4 is thus a beam path, are combined in the illumination beam path and detection beam path. This applies both with regard to the laser scanning module 9 and also with regard to the wide-field detection beam path 8, since, as will be explained later, illumination radiation is coupled into the switching mirror 11, which together with the wide field detection beam path 8, d. H. realized the CCD detector 6 microscopy.

The switching mirror 1 1 and the beam splitter 4 are combined to form a beam splitter module 12, which makes it possible to change the switching mirror 1 1 and the beam splitter 4 depending on the application. This is also illustrated by double arrows. Next in the beam splitter module 12, an emission filter 13 is provided, which is located in the wide-field detection beam path 8 and the spectral components, which can propagate through the wide-field detection beam 8, appropriately filters. Of course, the emission filter 13 in the beam splitter module 12 is interchangeable. The laser scanning module 9 receives laser radiation required for operation via an optical fiber 14 from a laser module 15. In the construction shown in FIG. 1, a collection illumination beam path 16 is coupled in at the beam splitter module 12, more precisely at the switching mirror 14, through which illumination radiation for different microscopy methods runs. In this collective illumination beam path 16 different illumination beam paths of individual lighting modules are coupled. For example, a wide-field illumination module 17 couples via a switching mirror 18 wide-field illumination radiation in the collection illumination beam path 16, so that the tube 2 is far-field illuminated via a tube lens 27 and the lens 3. The wide-field illumination module can have, for example, an HBO flap. As a further illumination module, a TI RF illumination module 19 is provided, which realizes a TIRF illumination with a suitable position of the switching mirror 18. The TI RF illumination module 19 receives radiation from the laser module 15 via an optical fiber 20. The TI RF illumination module 19 has a mirror 21 which is longitudinally displaceable. As a result of the longitudinal displacement, the illumination beam emitted by the TI RF illumination module 19 is displaced perpendicular to the main propagation direction of the emitted illumination beam, as a result of which the TI RF illumination is incident on the objective 3 at an adjustable angle to the optical axis of the objective. In this way, simply the necessary angle of total reflection on the cover glass can be ensured. Of course, other means are suitable for effecting this angular adjustment. Furthermore, the illuminating beam path of a manipulator module 22 is coupled to the collecting illumination beam path, which likewise receives radiation from the laser module 15 via an unspecified optical fiber and leads a point or line-shaped beam distribution over the sample 2 in a scanning manner. The manipulator module 22 thus corresponds essentially to the illumination module of a laser scanning microscope, and consequently the manipulator module 22 can also be operated in combination with the detector of the laser scanning module 9 or the wide-field detection of the CCD detector 6.

In the collective illumination beam path 16, a grid 23 is further provided, the lattice constant is below the cutoff frequency, which can be transmitted with the microscope 1 in the sample 2. The grating 23 is displaceable transversely to the optical axis of the collective illumination beam path 16. For this purpose, a corresponding displacement drive 24 is provided.

Downstream of the grating in the illumination direction, an image field rotator 25, which is rotated by a rotator drive 26, is located in the collective illumination beam path 16. The field rotator may be, for example, an Abbe-König prism. The modules and drives and detectors of the microscope 1 are all connected via unspecified lines to a control device 28. This connection can be made for example via a data and control bus. The control device 28 controls the microscope 1 in different operating modes.

The controller 28 thus allows the microscope 1 classical microscopy, d. H. Wide Field Microscopy (WF), Laser Scanning Microscopy (LSM) and also fluorescence microscopy with total internal reflection (TIRF). The microscope of FIG. 1 essentially has two modules suitable for laser scanner illumination, namely the laser scanning module 9 and the manipulator module 22. Of course, other combinations are also possible. These modules are coupled via tube lenses with the objective 3 to the sample 2. The manipulator module 22 only contains the excitation part of a laser scanning module, without detection. As a result, the sample can be spot-illuminated and the illumination spot scanned over the sample 2. Preferably located in the manipulator module 2 and a switching unit, for. As a switching lens or cylindrical lens with which a switch between a punctiform and a linear illumination. This line-shaped illumination is particularly advantageous when the grating 23, which is located in an intermediate image of the collection illumination beam path 16, is pivoted in and lies perpendicular to the line of the line-shaped illumination.

As an alternative to the grating 23, a variably adjustable strip modulator or a DMD for producing a structured illumination in the sample 2 can also be used. Then, of course, the displacement drive 24 and the on / Ausschwenkbarkeit the grid 23 is no longer required.

The field rotator 25 allows the structured illumination generated by the grating 23 (or elements replacing it) to rotate about the optical axis of the collection illumination beam path 16 so that the structured illumination is at different angles in the sample 2 ,

For switching between individual operating modes, the switching mirrors 18 and 11 and the beam splitter 4 are set appropriately. For this purpose, folding or Einschenkspiegel can be used in the realization, so that a switch between the modes can be done sequentially. Alternatively, dichroic mirrors are also possible which allow simultaneous operation of the various modules.

The beam splitter 4 is preferably designed as a dichroic beam splitter, wherein the spectral properties are adjustable so that spectral components of fluorescence emission of Labeling molecules to be detected by means of the CCD detector 6, get into the wide-field detection beam path 8 and the remaining spectral components are transmitted as possible. In order to increase the flexibility with regard to the usability of marking molecules having different emission characteristics, a plurality of different beam splitters 4 and emission filters 13 are interchangeably arranged in the beam splitter module 12, e.g. B. on a filter wheel.

The microscope described above now serves to generate a high-resolution microscope image. For this purpose, the control unit 28 is suitably designed, for example by a suitable programming. Before discussing possible process sequences with reference to FIGS. 4 and 5, essential features and principles will first be explained with reference to FIGS. 2 and 3, which are components of the microscopy methods to be described.

Fig. 2 exemplifies the SIM concept. For this purpose, the sample microscopically microscoped in the microscope 1 of FIG. 1 is repeatedly field-imaged, wherein different illumination states are set for each image. A set 39 of individual images 40 is thus obtained. The images 40 differ with regard to a structuring 41 of the illumination which is applied to the sample by the illumination beam path 8. As can be seen, the structuring 41 is different in different images 40. Overall, as the set 39 shown in plan view in FIG. 2 shows, there are nine individual pictures 40; So there are nine different orientations of structuring 41. The different structuring are illustrated in the representation of FIG. 2 by an appendix ".01" to ".09" to the reference symbol 40 of the respective image. The plan view of the individual images 40 in FIG. 2 shows that the nine orientations differ with regard to a displacement position or a rotational position of the structuring 40. The number of nine is exemplary of the orientations used. It is also possible a higher number, as is known from the publications listed in the general part of the description of the principle SIM.

The individual images 40 of the sentence 39 are recorded successively, with the structuring 41 of the illumination being correspondingly displaced or rotated between the recording of each image 40.

From the entire set 39, a high-resolution image 43 is then generated by means of a Fourier transformation 42. This image 43 can, as will be explained below with reference to FIG. 4, serve as a raw image for a further high-resolution processing. Fig. 2 thus illustrates features of the SIM principle. However, the structuring 41 is to be understood as purely exemplary. In particular, it does not have to be a line structure. The schematically drawn lines along the line can also be further structured. Similarly, it is possible to use a scanned confocal point illumination with confocal detection instead of the linear illumination used in the aforementioned SIM publications, as described in the publication C. Müller and J. Enderlein, "Image scanning microscopy", Physical Review Letters, 104, 198101 (2010) This principle is referred to as ISM Of course, then there are not nine orientations of structured illumination, but a suitable plurality of frames obtained from scanning a sample a particular scan position, ie a certain grid state when scanning the image.

Fig. 3 shows schematically another principle which is used for high-resolution microscopy. According to this principle, a sequence 44 of images 45 is taken, and as explained above with reference to the SOFI concept, each image 45 contains a different flashing state of the fluorophores in the sample 2. That the sequence 44 is a time series is illustrated by the suffixes attached to the reference number 45 of the pictures. The individual pictures have the words ". T1" to ". Tn". From the sequence 44, a high-resolution raw image 47 is again generated by means of a processing which uses a cumulant function 46. The n images 45 are thus converted into a single image 47.

This being said, a first method for high-resolution microscopy will now be explained with reference to FIG. For this purpose, sentences 39 are generated repeatedly, each consisting of images 40. o1 to 40. o9. Through this repetition there are a total of n sentences 39.t1 to 39.tn. In each sentence, the necessary for the SIM concept orientations of the illumination states are present, which should be clarified by the reference of the reference numerals of Fig. 2 in Fig. 4. The same applies of course in the event that not the SIM principle, but the ISM principle is used.

Now, by using Fourier filtering 42 from each set 49.t1 to 49.tn, a sequence 50 of raw pictures 49.t1 to 49.tn is generated. These raw images 49 are temporally spaced and, due to the use of the SIM principle, contain image information with a higher resolution than they provide the microscope a priori. Using the cumulant function 46, the sequence 50 is now converted into a high-resolution image 51. Its resolution is higher than the resolution in the raw images 49. In a second microscopy method, a sequence 54 of images 45 is recorded for each orientation .o1 to .o9, so that a separate sequence exists for each orientation. Each sequence is then converted using the cumulant function 46 into a high-resolution raw image, so that a total of 54 sets of raw images 53. o1 to 53. o9 is given. The set 54 thus contains raw images 53 in the different illumination structures required for the SIM principle. The same applies in turn to the application of the ISM principle. The set 54 of raw images is then converted using the Fourier filtering 42 into a high-resolution image 55 whose spatial resolution is higher than that of each raw image 53 whose resolution is higher again than the optics of the microscope 1 actually permits due to the physical limitations ,

Claims

claims 1 . Microscopy method for generating a high-resolution image (55) of a sample (2), the method comprising the following steps: a) the sample (2) is provided with a marker which gives off statistically flashing luminescent radiation after excitation, or a sample (2) is used which distributes locally after excitation, emitting statistically flashing luminescent radiation, b) the sample (2) is excited to luminescence by structured illumination, whereby the sample is repeatedly illuminated in different illumination states (.o1 -.o9) of the structured illumination, c) the luminescent sample (2) is repeatedly imaged onto a detector in each of the different illumination states so that an image sequence (44.o1 -44.o9) is obtained for each of the different illumination states (.01-.9), d) is generated from each image sequence (44.o1 -44.o9) by means of a cumulant function (46) which evaluates intensity fluctuations in the image sequence caused by the flashing, a raw image having an increased spatial resolution beyond the optical resolution of the image such that a high-resolution raw image (53.o1 -53.o9) is obtained for each of the different illumination states (.o1 -.o9), e) from the obtained raw images (53.o1 -53.o9) by a computational processing (42) comprising a Fourier filtering, the high-resolution image (55) having a spatial resolution which is increased compared to the raw images (53.o1 -53.o9), is produced. 2. A microscopy method for producing a high-resolution image (51) of a sample (2), the method comprising the following steps: a) the sample (2) is provided with a marker which gives off statistically flashing luminescent radiation after excitation, or a sample (2) is used which distributes locally after excitation, emitting statistically flashing luminescent radiation, b) the sample (2) is excited to luminescence by structured illumination, the sample being repeatedly illuminated with a set of different illumination states (.o1 -.o9) of the structured illumination, c) the luminescent sample (2) is imaged onto a detector in each set and there in each of the different illumination states so that a sequence of sets (39.t1 -39.tn), each for each different illumination state (.o1 -. o9) comprising an image (40) is obtained, d) from each set (39.t1 -39.tn) by a computational processing (42) comprising a Fourier filtering a raw image (49.t1 -49.tn) is generated with spatial resolution increased beyond the optical resolution of the image, so that a sequence (50) of raw images (49.t1 -49.tn) is obtained, e) from the sequence (70) of raw images (49.t1 -49.tn) by means of a cumulant function (46), which evaluates intensity fluctuations caused by the flashing in the sequence (50), the high-resolution image (51), the one opposite the raw images (49.t1 -49.tn) increased spatial resolution is generated. A method according to claim 2, wherein a marker or sample is used which emits blinking luminescent radiation at a medium blink rate, and in step c) the recording duration of each frame is chosen such that the time for generating a set (39. t1 -39.tn) is not greater than the reciprocal of the average flash rate, preferably not greater than 1/10 of the reciprocal. 4. The method of claim 1, 2 or 3, wherein the different illumination states have three rotational positions and per rotational position at least three shift positions of the structured illumination and the sample is imaged on a pixel surface detector. 5. Microscope for generating a high-resolution image (55) of a sample (2), the sample (2) is provided with a marker, the randomly flashing after excitation Emits luminescence radiation, or a sample that distributes locally after excitation, emitting statistically flashing luminescent radiation, wherein the microscope has:  a detection beam path (8) and an illumination beam path (16), wherein the Detection beam (8) images the sample (2) on a detector (6) and the illumination beam path (16) the sample (2) illuminated by structured illumination for excitation of luminescence, wherein the illumination beam path (16) means to Realization of different illumination states (.o1 -.o9),  a computing and control device (28) which controls the microscope (1) so that the Sample (2) repeatedly illuminates the different illumination states (.o1-.o9) of the structured illumination, the luminescent sample in each of the different ones Illumination states (.o1 -.o9) is repeatedly imaged onto a detector, so that an image sequence (44.o1 -44.o9) is obtained for each of the different illumination states (.o1 -.o9), the arithmetic and control device (28) from each image sequence (44.o1 -44.o9) by means of a cumulant function (46), which evaluates the intensity fluctuations in the image sequence caused by the flashing, a raw image, one beyond the optical resolution of the image has increased spatial resolution, so that a high-resolution raw image (53.o1 -53.o9) is obtained for each of the different illumination states (.o1 -.o9),  the arithmetic and control device (28) from the obtained raw images (53.o1 -53.o9) by a computational processing (42) comprising a Fourier filtering the high-resolution image (55), the one compared to the individual images (53.o1 -53. o9) has increased spatial resolution. 6. Microscope for generating a high-resolution image of a sample (2), the sample (2) is provided with a marker that emits randomly flashing luminescence after excitation, or a sample that distributes locally after excitation, statistically flashing emitting luminescence, wherein the Microscope has: a detection beam path (8) and an illumination beam path (16), wherein the detection beam path (8) images the sample (2) onto a detector (6) and the illumination beam path (16) images the sample (2) with structured illumination to excite luminescence illuminated, wherein the illumination beam path (16) has a device for the realization of different illumination states (.o1 -.o9), - a computing and control device (28) which controls the microscope (1) so that the sample (2) is repeatedly illuminated with a set of different illumination states (.o1 -.o9) of the structured illumination, and the luminescent sample in each set and there in each of the different illumination states (.o1 -.o9) is imaged onto a detector (6), so that a sequence of sets, each comprising one image per different illumination state, is obtained,  the arithmetic and control device (28) generates a raw image (49.t1 -49.t2) from each set by means of a computational processing (42) comprising a Fourier filtering with spatial resolution increased beyond the optical resolution of the image, so that a sequence (50 ) of raw images (49.t1 -49.tn), - The arithmetic and control device (28) from the sequence (50) of raw images (49.t1 -49.tn) by means of a cumulant function (46), which evaluates caused by the flashing intensity fluctuations in the sequence (50), the high-resolution image (55) which has a spatial resolution which is increased compared to the raw images (49.t1 -49.tn). A microscope according to claim 6, wherein the sample (2) or one of its markers emits flashing luminescent radiation at a medium blink rate, and the recording duration of each image is set so that the time for generating a sentence (39.t1 -39.tn) is not greater than the reciprocal of the average flash rate, preferably not greater than 1/10 of the reciprocal. 8. A microscope according to claim 5, 6 or 7, wherein the different illumination states have three rotational positions and per rotational position at least three displacement positions of the structured illumination and the detection beam path (16) the sample (2) on a pixel having surface detector (6).